Biological Methods for Cell-Cycle Synchronization of Mammalian Cells

Davis, Penny K.; Ho, A. D.; Dowdy, Steven F.

doi:10.2144/01306rv01

Cited by 188 publications

(161 citation statements)

References 63 publications

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“…Cell Synchronization-BHK cells were synchronized by serum deprivation as described previously (36). Briefly, 10 T-75 flasks of exponentially growing cells (60 -80% confluent) were serum-starved in DMEM, 0.1% fetal bovine serum for 24 h. The cells were then stimulated by the addition of 25 ml of DMEM supplemented with 10% fetal bovine serum and incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

Sigoillot

Berkowski

Sigoillot

et al. 2003

Journal of Biological Chemistry

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De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be upregulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent downregulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G 1 , the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G 1 . Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.The activity of the de novo pyrimidine biosynthetic pathway closely parallels the growth rate of the cell and is highest during periods of rapid proliferation (1-13). Cells growing in culture synthesize pyrimidines at a rapid rate during the exponential growth phase, but the pathway is much less active when the cells enter the stationary phase or have their growth arrested by serum deprivation (14 -18). In an earlier study, Mitchell and Hoogenraad (19) show that pyrimidine biosynthesis is maximally activated during the S phase of the cell cycle. Thus, the de novo biosynthetic pathway plays a dominant role in providing precursors for the synthesis of DNA that accompanies cell division. Furthermore, there is strong evidence (7,11,12) that the activation of the pathway is a prerequisite for the proliferation of tumor and neoplastic cells.The flux through the pathway is governed (20, 21) by the activity of carbamoyl-phosphate synthetase II (CPSase), 1 a component of CAD (Fig. 1), the multifunctional protein (22-24) that init...

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Section: Methodsmentioning

confidence: 99%

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

Sigoillot

Berkowski

Sigoillot

et al. 2003

Journal of Biological Chemistry

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“…Drugs such as actinomycin C 13 can be used to arrest mitosis at various stages, but the dosage must be carefully controlled to avoid apoptosis or other toxic effects which may affect cell migration. Serum starving 32 is the most common non-pharmaceutical method for minimizing proliferation in wound healing assays, Figure 1. Images from a scratch assay experiment at different time points.…”

Section: Sample Preparation: Cell Culturementioning

confidence: 99%

An introduction to the wound healing assay using live-cell microscopy

Jonkman

Cathcart

et al. 2014

Cell Adhesion & Migration

537

348

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“…Ideally, the growth state of the culture prior to synchronization should be clearly defined, and the technique employed should not perturb the cells from that state. Most synchronization procedures are initiated on cells presumed to be growing exponentially in a culture flask or bottle (Helmstetter 1969;Grdina et al 1984;Knehr et al 1995;Merrill 1998;Davis et al 2001). However, attainment of true exponential growth in batch cultures of mammalian cells, which may increase only about 10-fold in cell concentration from inoculation to early stationary phase, is problematic (e.g., Skehan and Friedman 1984).…”

Section: Introductionmentioning

confidence: 99%

“…Methods designed to obtain synchronously growing cultures by selecting cells of the same age or size from a growing culture are inherently less likely to disturb cell growth and cell cycle properties. Mitotic shake-off has been considered to be the least disturbing and easiest method for obtaining modest numbers of synchronous cells (Terasima and Tolmach 1963), as long as the state of the cells is not altered by transfer into fresh medium or treatment with mitotic inhibitors to increase cell yields (Merrill 1998;Davis et al 2001). …”

Section: Introductionmentioning

confidence: 99%

Technology for cell cycle research with unstressed steady-state cultures

et al. 2006

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A culture system for performing cell cycle analyses on cells in undisturbed steady-state populations was designed and tested. In this system, newborn cells are shed continuously from an immobilized, perfused culture rotating about the horizontal axis. As a result of this arrangement, the number of newborn cells released into the effluent medium each generation is identical to the number of cells residing in the immobilized population, indicating that one of the two new daughter cells is shed at each cell division. Thus, the immobilized cells constitute a continuous, steady-state culture because the concentrations, locations and microenvironments of the cells in the culture vessel do not vary with time. In tests with mouse L1210 lymphocytic leukemia cells, about 10 8 newborn cells were produced per day. This new culture system enables a multiplicity of cell cycle analyses on large numbers of cells assured to be from populations in steady-state growth.

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Biological Methods for Cell-Cycle Synchronization of Mammalian Cells

Cited by 188 publications

References 63 publications

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

An introduction to the wound healing assay using live-cell microscopy

Technology for cell cycle research with unstressed steady-state cultures

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